The blood-brain barrier (BBB) is a specialized physical and enzymatic barrier that segregates the brain from systemic circulation. The physical portion of the BBB is composed of endothelial cells arranged in a complex system of tight junctions which inhibit any significant paracellular transport.
The BBB functions as a diffusion restraint selectively discriminating against substance transcytosis based on lipid solubility, molecular size and charge thus posing a problem for drug delivery to the brain. Drug delivery across the BBB is further problematic due to the presence of a high concentration of drug efflux transporters (e.g., P-glycoprotein, multi-drug resistant protein, breast cancer resistant protein). These transporters actively remove drug molecules from the endothelial cytoplasm before they even cross into the brain.
The methods that are currently employed for drug delivery in treatment of brain diseases are generally non-specific, inefficient, complex to perform and very expensive.
An additional problem to consider when treating brain diseases is the diffusion of the drug on its vehicle across the tumor or affected tissue. Mostly the size, as well as other physiologic characteristics of the vehicles that are currently in use for such delivery of drugs to the brain, hamper efficient diffusion of the drug through the diseased tissue. The lack of efficient drug diffusion affects the efficacy of the treatment.
Peptides have been extensively studied as carrier molecules for drug delivery to the brain in hope they could be employed as drug delivery vehicles. Peptides, are, however, problematic due to their limited bioavailability. Even though methods to increase the bioavailability of such molecules have been intensively explored, they resulted in modest success at best.
Largely due to the strict regulation of molecular transport across the BBB and lack of success with currently available drug administration methods, a growing number of brain disorders, particularly cancers, remain treated inefficiently or not treated at all. There is currently a need for specific, less invasive and more efficient methods of drug delivery to the brain for the ever growing number of brain illnesses, particularly brain cancers.
Increased cell proliferation and growth is a trademark of cancer. The increase in cellular proliferation is associated with high turnover of cell cholesterol. Cells requiring cholesterol for membrane synthesis and growth may acquire cholesterol by receptor mediated endocytosis of plasma low density lipoproteins (LDL) (Brown, M. S. et al., Science 232:34-47 (1986)), the major transporter of cholesterol in the blood, or by de novo synthesis. LDL is taken up into cells by a receptor known as the LDL receptor (LDLR); the LDL along with the receptor is endocytosed and transported into the cells in endosomes. The endosomes become acidified and this releases the LDL receptor from the LDL; the LDL receptor recycles to the surface where it can participate in additional uptake of LDL particles (Maletinska, L. et al., Cancer Res 60:2300-2303 (2000)).
There is a body of evidence that suggests that tumors in a variety of tissues have a high requirement for LDL to the extent that plasma LDLs are depleted. The increased import of LDL into cancerous cells is thought to be due to elevated LDL receptors (LDLR) in these tumors. Some tumors known to express high numbers of LDLRs include some forms of leukemia (Vitols, S. et al, Blood63:1186-1193 (1984); Vitols, S. et al., Lancet2:1150-1153 (1985), 9), lung tumors (Vitols, S. et al., Cancer Res 52:6244-6247 (1992); Lundberg, B. Cancer Res 47:4105-4108 (1987)), colorectal tumors (Lum, D. F. et al., Int J Cancer 83:162-166 (1999)) and ovarian cancer (Avall-Lundqvist, E. H. et al., Acta Oncol 35:1007-1010 (1996)).
Comparative studies of normal and malignant brain tissues have shown a high propensity of LDLRs to be associated with malignant and/or rapidly growing brain cells and tissues. Using immunohistochemistry, Pitas et al. (Pitas, R. E. et al., J Biol Chem 262:14352-14360 (1987)) examined monkey and rat brain and reported relatively few LDLRs in normal neurons and glial cells. Limited staining for LDLR was shown in astrocytes abutting the arachnoid space and in astrocytes in the white matter. However, large numbers of high affinity LDLR have been reported for the rapidly proliferating primary glial cells isolated from 1-2 day old rat pups (Jung-Testas, I. et al., J Steroid Biochem Molec Biol 42:597-605 (1992)). These findings strongly suggest that rapidly growing brain cells such as those seen in early development and in aggressively growing brain tumors exhibit increased expression of LDLRs due to their increased requirement for cholesterol.
Additional in vivo studies showed that LDLRs do appear in brain malignancies. Leppala et al. (Leppala, J. et al., Br J Cancer 71:383-387 (1995)) used PET imaging, and demonstrated that 99mTc-LDL localizes in human brain tumors in vivo but not in normal brain.
Although the major transporter of cholesterol into cells and tissues, LDL is excluded from performing this function in the brain being too large to cross the blood brain barrier.
The LDLR ligand is apoB100, a 514 kDa glycoprotein on the surface of LDL. This lipid binding protein is very large (˜500 kD) and consists of hydrophobic domains, amphipathic beta sheets and amphipathic helices; unlike other apolipoproteins it is not an exchangeable protein. The protein is highly insoluble in aqueous medium and has a propensity to aggregate thus making it a difficult protein with which to work. The structure of apoB100 is now well defined (Segrest J. P. et al., J Lipid Res 42:1346-1367 (2001)). Early work of Yang et al. (Yang C.-Y. et al., Nature 323:738-742 (1986)) suggested that the apoB sequence between a.a. 3345-3381 contains the LDLR binding domain. Indeed, Yang et al. showed that a synthetic peptide corresponding to this region can bind to human skin fibroblasts and regulate HMG-CoA reductase. Law and Scott (Law A. et al., J Lipid Res 31:1109-1120 (1990)), using cross-species comparisons, further refined the binding domain to a nine amino acid segment consisting of residues 3359-3367. The results of these studies were further confirmed by site directed mutagenesis studies of Boren et al. (Boren, J. et al., J Clin Invest 101:1084-1093 (1998)) who showed that apoB100 protein with mutations in this region lacked the ability to interact with the LDLR.
Even though human cerebral spinal fluid (CSF) contains apolipoproteins including apoE, apoA-I, apoC-III and C-II (Roheim, P. S. et al., Proc Natl Acad Sci USA 76:4646-4649 (1979)) thus making a case for lipid transport in the brain and cholesterol homeostasis similar to that of other tissues, apoB100 was not detected in the CSF consistent with the exclusion of LDL by the blood brain barrier. CSF lipoprotein particles examined by electron microscopy were in the size range of 11-13 nm.
Among the problematic and inefficiently treated brain cancer is glioblastoma multiforme (GBM). This devastating brain tumor is 100% fatal. Moreover, over 85% of total primary brain cancer-related deaths are due to GBM. Current therapies rely on a multimodal approach including neurosurgery, radiation therapy and chemotherapy. Even the best efforts using these approaches have resulted in only a modest increase in survival time for patients afflicted with this tumor.
GBM being gliomas of the highest malignancy are characterized by uncontrolled, aggressive cell proliferation and general resistance to conventional therapies. GBM cells in culture have high numbers of low density lipoprotein receptors (LDLR) (Maletinska, L. et al., Cancer Res 60:2300-2303 (2000)). Since this receptor is nearly absent in neuronal cells and normal glial cells, it represents an ideal target for the delivery of therapeutic agents such as cytotoxins or radiopharmaceuticals. Efforts to improve existing therapies or to develop new ones have not been successful and the outcome of treatment for malignant gliomas is only modest, at best, with a median survival time of approximately 10 months (Miller, P. J. et al., Int J Radiat Oncol Biol Phys 19:275-280 (1990); Shibamoto, Y. et al., Radiother Oncol 18:9-17 (1990); Barker, F. G. 2nd et al., Neurosurgery 42:981-987, (1998)).
Unlike normal brain cells that have few LDL receptors, GBM cells in culture have high numbers of LDL receptors on their surface (Brown, M. S. et al., Science 232:34-47 (1986)). Other brain cancers are likely to also have high expression of LDLR due to the highly proliferative nature of the cancerous tissue and need for cholesterol turnover.
This suggests that the LDL receptor is a potential unique molecular target in GBM and other brain malignancies for the delivery of anti-tumor drugs via LDL particles.
The present invention addresses the need of targeted delivery of therapeutic compounds to cancers and other diseases where the LDLR is presented on the cell surface via a synthetically synthesized LDL nanoparticle capable of carrying and transporting therapeutics.